Long-term Objectives: Our goal is to develop therapy against transforming growth factor-beta (TGFb) to break the vicious cycle of bone metastasis. TGFbeta in the bone microenvironment dramatically changes the phenotype of metastatic tumor cells, causing them to express factors that stimulate pathological changes in the skeleton. We hypothesize that TGFb signaling in tumors is activated by the bone microenvironment, acts through the Smad pathway in tumor cells and can synergize with hypoxia to drive the vicious cycle. Inhibiting both TGFb and hypoxia pathways will provide more effective treatment of bone metastases than single agents. Specific Aims: Aim 1 will test inhibitors that act on different targets in the TGFb pathway: Smad2/3, Smad7 and p38MAP kinase. Aim 2 will test if bone metastases are increased by standard sex-steroid ablation treatments for breast and prostate cancer, which stimulate bone resorption and can increase TGFb in the bone microenvironment. Aim 3 will test whether hypoxia increases TGFb signaling in tumor cells both at the molecular level in vitro and in bone metastases in vivo. Aim 4 will use new imaging procedures to demonstrate that TGFb signaling is directly and specifically activated when tumors grow in bone. Health-relatedness: We will use animal models of human breast cancer, prostate cancer and melanoma. Experiments use drugs that are already approved or in clinical development. Research Design & Methods: Three tumor types will be grown as xenografts in immunodeficient mice. Mice will be treated with well-characterized small-molecule inhibitors of TGFb, Smads, p38MAPK, hypoxia, and bone resorption. Tumor and bone endpoints will be evaluated by X-radiography, bioluminescent imaging, histology, and quantitative bone histomorphometry. MicroPET imaging of gene-reporter activity will provide an accurate readout of activation of TGFb signaling in tumors growing in the bone microenvironment. In vivo studies will be complemented by detailed molecular studies on actions of the TGFb-inhibitory Smad7 and the corepressors Ski & SnoN and effects on TGF-beta1 and Smad7 promoters. Rationale & Techniques: Animal models permit rapid preclinical testing of single agent and combination therapies to prevent and treat bone metastases and provide statistically significant endpoints of tumor growth and bone responses. Imaging of tumor in bone permits direct validation in vivo of mechanisms that are inaccessible by other means. Relevance to Public Health: The majority of patients who die from breast or prostate cancer have bone metastases, which cause severe bone pain, fractures, hypercalcemia and nerve compression. Bone metastases are incurable. We will test a series of five drugs and combinations to design better therapy to treat and cure bone metastases. The drugs are chosen to be specific against bone metastases. All are of low toxicity, and all but one is already approved or in clinical trials and preclinical development.